Sulfated lyso-ganglioside derivatives

ABSTRACT

Semisynthetic analogues of gangliosides selected from the group consisting of N-sulfo-, N-hydrocarbyl-sulfonyl- and N-hydrocarbyloxy-sulfonyl-N,N&#39;-dilyso-gangliosides and the N&#39;-acyl derivatives thereof, N&#39;-sulfo-, N&#39;-hydrocarbylsulfonyl- and N&#39;-hydrocarbyloxy-sulfonyl-N,N&#39;-dilyso-gangliosides and the N-acyl derivatives thereof, N,N&#39;-di or polysulfo-N,N&#39;-di- or poly-lyso-gangliosides, N,N&#39;-di- or polyhydrocarbylsulfonyl-N,N&#39;-di- or poly-lyso-gangliosides and N,N&#39;-di- or polyhydrocarbyloxy-N,N&#39;-di- or poly-lyso-gangliosides, and functional derivatives thereof, and salts of these compounds, have protective activity against neurotoxicity induced by excitatory amino acids and are foreseen to be used in therapy in the nervous system and in modulating of the expression of determinants such as CD 4  on the surface of human cells in the immune system.

This is the U.S. National stage entry under 35 U.S.C. 371 ofPCT/US94/01966, filed Mar. 4, 1994.

OBJECT OF THE INVENTION

The present invention concerns novel semisynthetic gangliosideanalogues, and more precisely N-sulfo-, N-hydrocarbylsulfonyl- andN-hydrocarbyloxysulfonyl-N,N'-dilyso-gangliosides and the N'-acylderivatives thereof, N'-sulfo-, N'-hydrocarbylsulfonyl- andN'-hydrocarbyloxysulfonyl-N,N'-dilyso-gangliosides and the N-acylderivatives thereof, N,N'-di or polysulfo-N,N'-di- orpoly-lyso-gangliosides, N,N'-di or polyhydrocarbylsulfonyl-N,N'-di orpoly-lyso-gangliosides and N,N'-di- orpolyhydrocarbyloxysulfonyl-N,N'-di or polylyso-gangliosides and thefunctional derivatives thereof, and salts of all these compounds.

The novel derivatives have interesting pharmacological properties,especially protective activity against neurotoxicity induced byexcitatory amino acids, such as glutamic acid, and can therefore be usedin therapy for the nervous system, such as for conditions followingdegeneration or lesions, i.e., ischemia, hypoxia, epilepsy, trauma andcompression, metabolic dysfunction, aging, toxic-infective diseases andchronic neurodegeneration, such as Alzheimer's disease, Parkinson'sdisease or Huntington's chorea.

The novel compounds of the invention, thanks to their neuritogenicactivity, can be used to advantage in therapies aimed at nervousfunction recovery, such as in peripheral neuropathies and pathologiesassociated with neuronal damage.

Moreover, the novel derivatives which are object of the presentinvention have valuable properties for the modulation of the expressionof specific determinants, such as CD₄, present on the surface of humancells belonging to the immune system.

The ability of the above compounds to modulate expression of themolecule CD₄, a membrane glycoprotein expressed in various cell typessuch as thymocytes, lymphocytes, monocytes and macrophages has greatapplicative potential in a wide range of human pathologies.

Compounds of the present invention may have therapeutic use in allsituations wherein it is necessary to prevent and/or treat infectionsinvolving CD₄₊ cells, such as infections the etiological agents whereofare microorganisms belonging to the human immunodeficiency (HIV) familyof viruses. Moreover, the modulation of CD₄ is useful in systemic ororgan-specific autoimmune diseases, such as multiple sclerosis,rheumatoid arthritis, chronic polyarthritis, lupus erythematosus,diabetes mellitus, and also to prevent the phenomenon of organtransplant rejection as well as rejection by the transplanted materialagainst the host, as in the case of bone marrow transplant, and in allcases where the desired effect is to obtain tolerance towards "self" and"non-self" antigens.

Functional derivatives of the abovesaid semisynthetic gangliosideanalogues are for example esters and amides of the carboxyl groups ofsialic acid residues and may also be inner esters with lactone bondsbetween their sialic carboxyl groups and hydroxyl groups in theoligosaccharide, analogous to those known in the case of gangliosides,and possibly also the derivatives of all these compounds, in which thehydroxyl groups are esterified with organic acids or sulfuric acid.

Of particular interest are the esters with organic acids wherein all thehydroxyl groups, both those of the saccharide component and the freegroups of sialic acid, and the hydroxyl group of the ceramide residueare esterified, that is "peracylated" and also the persulfatedderivatives, i.e. those wherein all the hydroxyl groups are esterifiedwith sulfuric acid.

The term "N,N'-dilyso-ganglioside" in the aforesaid definition means aganglioside from which both the acyl group on the amino group in itsceramide residue, and the acyl group(s) of its sialic acid residue(s)have been removed. In the new compounds of the invention the ceramideamino group (position N) or all the amino groups of sialic acid (aposition which is collectively called N' for brevity), or both the freeamino functions of N,N-dilyso-gangliosides are therefore acylated withthe above-mentioned radicals.

The term "-di-" therefore refers to positions N and N' in the sensedefined above and not to the actual number of deacylated aminofunctions.

In the expressions N'-sulfo, N'-hydrocarbyloxy-sulfonyl andN'-hydrocarbylsulfonyl-N,N'-dilyso-gangliosides the substituents inposition N' are equal in number to the neuraminic acids present in theganglioside. Therefore, the definition "di-" or "poly-" has been chosenfor the possible substituents in this position.

As explained below, the two groups substituting the N and N' aminogroups may be different, thus leading to "unsymmetrical" compounds.

In the substituted derivatives in just one of the positions N and N',with one of the sulfured radicals indicated previously, the other aminogroup may be substituted with an acyl group of an organic acid.

In the illustrative examples and also elsewhere the expression "-lyso"without any indication of the position, as in the literature, should betaken to mean the position of the ceramide amino group.

The salts can be metal salts of the free carboxyl groups, for examplethe sialic carboxyl groups or carboxyl groups possibly present in theacyl residues of the acyl amido groups, as for example when the acylgroup is derived from a polybasic acid, such as succinic acid; moreoverthe salts may be derived from organic bases, especially from those whichare therapeutically acceptable. In addition, metal salts or those withorganic bases on any sulfate groups which may be present may bementioned.

Another aspect of the invention is directed to salts with acids at aminogroups which may be present, for example at the amino group of aN-hydrocarbyl-sulfonyl-N,N'-dilyso-ganglioside. In this case too thesalts of acids which can be used in therapy are preferred. Also includedin the invention are salts deriving from metals, bases or acids notnormally used in therapy and such salts may possibly be used for thepurification of the new products.

The aforesaid semisynthetic ganglioside analogues are novel.

Another aspect of the invention is directed to the use of these novelcompounds in therapy, especially to treat the abovementioned disordersaffecting the central or peripheral nervous systems or the immunesystem. Moreover, the invention is directed to pharmaceuticalpreparations containing one or more of the novel compounds, possiblytogether with a pharmaceutical excipient or vehicle.

The term "hydrocarbyl" as used alone or as a part of a hydrocarbyloxymoiety in the abovesaid definition of the new compounds indicates themonovalent residue of a substituted or unsubstituted, saturated orunsaturated hydrocarbon, i.e. the radical obtained from the formula of ahydrocarbon by elimination of a hydrogen atom, and such radicals aretherefore, for example, alkyl, aryl, aralkyl or cycloalkyl. The alkylgroups have preferably a maximum of 24 carbon atoms and are preferablyC₁₋₆ alkyls, especially methyl, ethyl, propyl, isopropyl, n-butyl,isobutyl or n-pentyl or trimethylmethyl. An aryl radical has preferablya maximum of 12 carbon atoms and is preferably a phenyl group, possiblysubstituted with 1-3 C₁₋₄ alkyl groups or C₁₋₄ alkoxy groups, especiallymethyl or methoxy groups. An aralkyl group has preferably a maximum of12 carbon atoms, the aliphatic part being preferably a C₂₋₄ alkylenegroup, and the aromatic part is the same group already indicated aspreferential for the aromatic groups.

Cycloalkyl is preferably a radical deriving from cyclopropane,cyclobutane, cyclopentane or cyclohexane, or from one of theirhomologues substituted with C₁₋₄ alkyl groups, for example methylgroups.

The hydrocarbyl groups can also be substituted with functions,especially by hydroxyl or amino groups or halogens, for example chlorineor bromine atoms. Moreover, the hydrocarbyl groups may also beinterrupted in the carbon atom chain by heteroatoms, such as especiallyby nitrogen atoms or --NH-- groups, respectively. Unsaturatedhydrocarbyl groups are especially those deriving from long-chained alkylgroups, for example C₁₄₋₂₂ alkenyl groups. The saturated alkyl radicalswith the same number of carbon atoms are also interesting.

Among the hydrocarbyl radicals of the aromatic series, special mentionshould be made of the phenyl variety with a maximum of 12 carbon atoms,for example the unsubstituted phenyl radical or a phenyl radicalsubstituted with between 1 and 3 substituents selected from the groupconsisting of C₁₋₄ alkyl groups and C₁₋₄ alkoxy groups, especiallymethyl and methoxy groups, amino groups and halogens such as chloro orbromo.

Araliphatic radicals, i.e. aralkyl radicals, are preferably those withthe same aromatic groups as those illustrated above for the aromatics,and with a C₂₋₄ alkylene radical in the aliphatic part.

Cycloalkyl hydrocarbyl radicals are preferably those with one singlering and with a maximum of 10 carbon atoms, especially cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl and their derivatives substitutedwith between 1 and 3 C₁₋₄ alkyl groups, especially methyl groups.

The carbon atom chain may be interrupted by heteroatom links thusleading to heterocyclic radicals such as pyridyl.

In the novel compounds of the present invention, wherein, besides Etsulphurated group in one of the positions N or N', there is an acylgroup in the other, the acyl group may be for example one of the groupspresent in natural gangliosides, i.e. an acyl deriving from a saturatedor unsaturated fatty acid with between 16 and 22 carbon atoms or from acorresponding hydroxy acid, as far as the amino group of the ceramideresidue is concerned, and an acyl deriving from acetic or glycolic acidwith regard to the amino group or the amino groups of the neuraminicresidue.

Also coming within the scope of the present invention are mixtures ofthe aforesaid chemical compounds, and in particular the derivatives ofnatural gangliosides obtainable by extraction from tissues of thenervous system or by enzymatic reactions, where the acyl groups aremixtures of acyl groups from higher aliphatic acids present at thesphingosine nitrogen atom and mixtures of acyl groups from acetic orglycolic acid at the neuraminic nitrogen and, wherein, possibly, hydroxygroups of the sialic acids are in esterified form.

The acyl groups present in position N or N' may derive from aliphaticacids and have preferably a maximum of 24 carbon atoms, especially thosewhich have between 12 and 16 carbon atoms and are straight-chained, orthose having between 1 and 11 carbon atoms and a straight or branchedchain, such as formic acid, acetic acid, propionic acid, butyric acids,valeric acids, especially n-valeric acid, isovaleric acid,trimethyl-acetic acid, caproic acid, isocaproic acid, heptanoic acid,octanoic acid, pelargonic acid, capric acid, undecanoic acid,di-tertbutyl-acetic acid and 2-propyl-valeric acid, as well as lauric,myristic, palmitic, oleic, elaidic and stearic acid.

The acyl radicals may also derive from aliphatic acids substituted withone or more polar substituents, such as halogens, in particularchlorine, bromine and fluorine, free or esterified hydroxy groups,ketone, ketal and acetal groups derived from lower aliphatic oraraliphatic alcohols, ketoxy or aldoxy or hydrazone groups, free oresterified mercapto groups with a lower aliphatic or araliphatic acid oretherified with lower aliphatic or araliphlatic alcohols, free oresterified carboxyl groups, free or esterified sulfon groups with loweraliphatic or araliphatic alcohols, sulfamide or sulfamide groupssubstituted with lower alkyl or aralkyl groups or lower alkyl groups,sulfoxide groups or sulfone groups derived from lower alkyl or aralkylgroups, or lower alkylene groups, nitril groups, free or substitutedamino groups, and ammonium derivatives of such amino groups.

The acyl radicals substituting one of the neuraminic or sphingosineamino groups of the new derivatives may also be radicals of an aromatic,araliphatic, cycloaliphatic, aliphatic-cycloaliphatic or heterocyclicacid.

Aromatic acyl groups are mainly those deriving from benzoic acid or itshomologues wherein the phenyl residue is substituted with, for example,1 to 3 C₁₋₄ alkyl or C₁₋₄ alkoxy groups, especially methyl and methoxygroups and/or by one of said polar groups, for example free or alkylatedamino groups.

Araliphatic acyl groups have preferably a C₂₋₄ alkylene chain as theiraliphatic portion, and the aromatic portion is preferably one of theabovesaid aromatic groups. Cycloaliphatic acyl radicals are preferablythose deriving from an alicyclic hydrocarbon with between 3 and 6 carbonatoms in the ring, such as cyclopropane, cyclobutane, cyclopentane andcyclohexane.

Heterocyclic radicals derive preferably from a monocyclic heterocycliccompound with just one heteroatom link, such as --O--, --N═, --NH--,--S--, and may be aromatic or aliphatic by nature, such as acids of thepyridine group, for example nicotinic or isonicotinic acid, the furanegroup, such as 2-furoic acid, or the thiophene group, such as2-thiophene-acetic acid, the imidazole group, such as 4-imidazole-aceticacid, or the pyrrole group, such as 1-methyl-2-pyrrole-carboxylic acid.

Functional derivatives of the new semisynthetic ganglioside derivativesaccording to the present invention are esters, inner esters and amidesof the sialic carboxylic groups. The ester groups derive particularlyfrom aliphatic alcohols and especially from those with a maximum of 12and preferably 6 carbon atoms, or from araliphatic alcohols withpreferably one single benzene ring, possibly substituted with 1-3 C₁₋₄alkyl groups, for example methyl groups, with a maximum of 4 carbonatoms in the aliphatic chain, or from alicyclic or aliphatic-alicyclicalcohols with one single cycloaliphatic ring and a maximum of 14 carbonatoms, or from heterocyclic alcohols with a maximum of 12 carbon atoms,and preferably 6, and one single heterocyclic ring containing aheteroatom link chosen from the group formed by --N═, --NH--, --O--, and--S--.

The amido groups of the carboxyl functions derive from ammonia or fromamines of any class, preferably with a maximum of 12 carbon atoms.Special mention should be made of lower aliphatic amines, such asmethylamine, ethylamine, propylamine, and butylamine.

Said alcohols and amines can then be substituted, especially byfunctions chosen from the groups formed by hydroxyl, amino, alkoxylgroups with a maximum of 4 carbon atoms in the alkyl moiety, carboxyl orcarbalkoxyl with a maximum of 4 atoms in the alkyl moiety, alkylamino ordialkylamino groups with a maximum of 4 carbon atoms in the alkyl group,and they may be saturated or unsaturated, especially with one doublebond. The alcohols may be monovalent or polyvalent, particularlybivalent. Of the aliphatic alcohols, special mention should be made ofthe lower alcohols with a maximum of 6 carbon atoms, such as methylalcohol, ethyl alcohol, propyl alcohol and isopropyl alcohol, n-butylalcohol, isobutyl alcohol, tert-butyl alcohol and, of the bivalentalcohols, ethylene glycol and propylene glycol. Of the araliphaticalcohols, special mention should be made of those with just one benzeneresidue, such as benzyl alcohol and phenethyl alcohol; of the alicyclicalcohols, preference is given to those with just one cycloaliphaticring, such as cyclohexyl alcohol (cyclohexanol) or terpene alcohols.Among the heterocyclic alcohols, special mention should be made oftetrafuranol and tetrapyranol.

Of the functional derivatives of the novel semisynthetic analoguesaccording to the present invention, special importance should be givento the peracylated derivatives on the hydroxyl groups of the saccharideportion of the sialic acids and ceramide.

In such derivatives the acyl radicals may derive from aliphatic,aromatic, araliphatic, alicyclic or heterocyclic acids, such as thoselisted above as acylating the N or N' acylamino groups. They arepreferably derived from aliphatic acids with a maximum of 10 carbonatoms and preferably 6 carbon atoms, such as formic, propionic,valerianic, butyric, caproic or hexanoic acid. They may also be derivedfrom substituted acids, preferably with the same number of carbon atoms,and particularly substituted with hydroxyl, amino, or carboxyl groups,such as lactic acid, glycine, malonic acid, maleic acid or succinicacid.

Among the acyl groups deriving from aromatic acids, particular mentionshould be made of those deriving from benzoic acid or its derivativessubstituted with methyl, hydroxyl, amino or carboxyl groups, such asp-amino-benzoic acid, salicylic acid or phthalic acid.

Of the esters corresponding to the ganglioside hydroxyls, specialconsideration should be given to the esters of sulfuric acid, above allthose wherein all the OH groups, namely those in the saccharide portion,the ceramide residue and in the sialic acid residues, are sulfated. SuchO-persulfated derivatives can be prepared from said semisyntheticanalogues by subsequent treatment with the sulfuric acidanhydride-trimethylamine complex, for example in dimethylformamide overa long period of time and at a high temperature.

Using shorter time intervals and/or smaller quantities of reagent it ishowever also possible to obtain partial esters of the basic ganglioside,and these too form part of the invention. Such partial esters usuallyrepresent mixtures with O-sulfated groups in various positions of theganglioside molecule.

The sulfated compounds are easily transformed into their metal ororganic base salts, for example into their alkali metal salts,especially sodium salts, by treatment with bases or basic salts, forexample, in the case of sodium salts, with sodium carbonate. Especiallyfor therapeutic applications, the sulfated esters are in the form ofsuch salts, especially sodium salts.

Among the most important basic gangliosides to be used in thepreparation of the novel derivatives are for example those wherein theoligosaccharide is formed by a maximum of 4 residues of saccharide, andwherein the saccharide portion is unitary. It is preferable to choosehexoses from the group formed by N-acetylglucosamine andN-acetylgalactosamine. The gangliosides of said group are for exampleextracted from vertebrate brains, such as those described in the article"Gangliosides of the Nervous System" in "Glycolipid Methodology", LloydA., Witting Fd., American Oil Chemists Society, Champaign, III, 187-214(1976) (see in particular FIG. 1), for example gangliosides GM₄, GM₃,GM₂, GM₁ -GlcNAC, GD₂, GD_(1a) -GalNAC, GT_(1c), G_(Q), GT₁, andparticularly those wherein the oligosaccharide contains at least oneglucose residue or one galactose residue and one N-acetylglucosamineresidue or N-acetylgalactosamine residue and above all the following:##EQU1## where Glc stands for glucose, GalNAC stands forN-acetylgalactose amine, Gal stands for galactose, NANA stands forN-acetylneuraminic acid.

One group of new derivatives according to the invention is representedby the following formulae, wherein only one of the R₃ substituentssignifies acyl. ##STR1## wherein: R=H or SO₃ H ##STR2## R₂ =--(CH₂)_(n)--CH₃, wherein n=12-14 R₃ =H, acyl or SO₂ R₅, provided that at least oneis SO₂ R₅ ##STR3## R₅ =alkyl, aryl or OX (X=H, alkyl, aryl)

Of the specific compounds of particular interest, special mention shouldbe made of the following derivatives of N-lyso GM₁ (ganglioside GM₁without its acyl group in the ceramide residue):

N-ethyl-sulfonyl-lyso-GM₁

N-propyl-sulfonyl-lyso-GM₁

N-n-butyl-sulfonyl-lyso-GM₁

N-n-pentyl-sulfonyl-lyso-GM₁

N-n-hexyl-sulfonyl-lyso-GM₁

N-n-heptyl-sulfonyl-lyso-GM₁

N-n-octyl-sulfonyl-lyso-GM₁

N-n-decyl-sulfonyl-lyso-GM₁

N-2-bromoethyl-sulfonyl-lyso-GM₁

N-hexadecyl-sulfonyl-lyso-GM₁

N-3-chloropropyl-sulfonyl-lyso-GM₁

N-6-bromohexyl-sulfonyl-lyso-GM₁

N-benzyl-sulfonyl-lyso-GM₁

N-4-chlorobenzyl-sulfonyl-lyso-GM₁

N-4-aminobenzyl-sulfonyl-lyso-GM₁

N-3,4,5-trimethoxybenzyl-sulfonyl-lyso-GM₁

N-sulfo-lyso-GM₁

Moreover, interesting compounds are derivatives sulfated at the hydroxygroups of each of the compounds specifically mentioned, which can bepartially sulfated derivatives containing for example just one sulfatedgroup (sulfo) on one of the saccharide hydroxyl groups and which aremixtures of various position isomers, or are persulfated derivatives,wherein all the hydroxyl groups in the ganglioside molecule areconverted into esters of sulfuric acid.

Also of interest are the derivatives corresponding to those which appearin the aforementioned list, deriving however from one of the followinggangliosides: GM₄, GM₃, GM₂, GD₂, GD_(1a), GT₁.

Of the derivatives of N'-lyso-GM₁ (i.e. ganglioside GM₁ without its acylgroup in the neuraminic residue), special mention should be made ofN'alkyl-sulfonyl-N'-lyso-GM₁, the alkyl of which is any one of the alkylgroups appearing in the above list for N-lyso-GM₁ derivatives, andN'aryl-sulfonyl-N'-lyso-GM₁, the aryl group of which is any one of thespecific groups of this type mentioned above and N'-sulfo-N'-lyso-GM₁and the esters thereof with alcohols deriving from any one of the alkylor aryl radicals featured in said N-alkyl or aryl-sulfonyl-GM₁derivatives, and the derivatives thereof partially or totally sulfatedat the hydroxyls groups. Of the derivatives of N,N'-dilyso-GM₁ (i.e.ganglioside GM₁ from which both acyl groups have been removed from theceramide and the neuraminic group) mention should be made of theN,N'-di-alky- and N,N'-di-aryl-sulfonyl-N,N'-di-lyso-GM₁, with an alkylor an aryl corresponding to the specific groups of this type which isfeatured in the above reported list of the N-alkyl- orN-aryl-sulfonyl-lyso-GM₁. Mention should also be made of the derivativeswherein one of the amino groups of ganglioside GM₁ is acylated with analkyl or arylsulfonic acid, wherein alkyl or aryl is one of theaforesaid specific groups in the list reported above for the derivativesof N-lyso-GM₁ and the other amino group is acylated with an acidselected from the group consisting of acetic acid, chloroacetic acid,dichloroacetic acid, propionic acid, n-valerianic acid, trimethylaceticacid, caproic and hexanoic acid, caprylic acid and undecylic acid, andin particular the di-sulfo derivative of N,N'-di-lyso-GM₁, and theesters thereof with alcohols deriving from any one of the alkyl or arylradicals featuring in the above list of derivatives of N-alkyl- orN-aryl-sulfonyl-lyso-GM1.

Thus, specific compounds are

N'-ethyl-sulfonyl-N'-lyso-GM₁

N'-propyl-sulfonyl-N'-lyso-GM₁

N'-n-butyl-sulfonyl-N'-lyso-GM₁

N'-n-pentyl-sulfonyl-N'-lyso-GM₁

N'-n-hexyl-sulfonyl-N'-lyso-GM₁

N'-n-heptyl-sulfonyl-N'-lyso-GM₁

N'-n-octyl-sulfonyl-N'-lyso-GM₁

N'-n-decyl-sulfonyl-N'-lyso-GM₁

N'-2-bromoethyl-sulfonyl-N'-lyso-GM₁

N'-hexadecyl-sulfonyl-N'-lyso-GM₁

N'-3-chloropropyl-sulfonyl-N'-lyso-GM₁

N'-6-bromohexyl-sulfonyl-N'-lyso-GM₁

N'-benzyl-sulfonyl-N'-lyso-GM₁

N'-4-chlorobenzyl-sulfonyl-N'-lyso-GM₁

N'-4-aminobenzyl-sulfonyl-N'-lyso-GM₁

N'-3,4,5-trimethoxybenzyl-sulfonyl-N'-lyso-GM₁

N'-sulfo-N'-lyso-GM₁

N,N'-di(ethyl-sulfonyl)-N,N'-dilyso-GM₁

N,N'-di(propyl-sulfonyl)-N,N'-dilyso-GM₁

N,N'-di-(n-butyl-sulfonyl)-N,N'-dilyso-GM₁

N,N'-di-(n-pentyl-sulfonyl)-N,N'-dilyso-GM₁

N,N'-di- (n-hexyl-sulfonyl)-N,N'-dilyso-GM₁

N,N'-di-(n-heptyl-sulfonyl)-N,N'-dilyso-GM₁

N,N'-di-(n-octyl-sulfonyl)-N,N'-dilyso-GM₁

N,N'-di-(n-decyl-sulfonyl)-N,N'-dilyso-GM₁

N,N'-di-(2-bromoethyl-sulfonyl)-N,N'-dilyso-GM₁

N,N'-di-hexadecyl-sulfonyl-N,N'-dilyso-GM₁

N,N'-di-(3-chloropropyl-sulfonyl)-N,N'-dilyso-GM₁

N,N'-di-(6-bromohexyl-sulfonyl)-N,N'-dilyso-GM₁

N,N'-di-benzyl-sulfonyl-N,N'-dilyso-GM₁

N,N'-di-(4-chlorobenzyl-sulfonyl)-N,N'-dilyso-GM₁

N,N'-di-(4-aminobenzyl-sulfonyl)-N,N'-dilyso-GM₁

N,N'-di-(3,4,5-trimethoxybenzyl-sulfonyl)-N,N'-dilyso-GM₁

N,N'-di-sulfo-N,N'-(dilyso-GM₁

N-ethyl-sulfonyl-N'-acyl-N,N'-dilyso-GM₁

N-propyl-sulfonyl-N'-acyl-N,N'-dilyso-GM₁

N-n-butyl-sulfonyl-N'-acyl-N,N'-dilyso-GM₁

N-n-pentyl-sulfonyl-N'-acyl-N,N'-dilyso-GM₁

N-n-hexyl-sulfonyl-N'-acyl-N,N'-dilyso-GM₁

N-n-heptyl-sulfonyl-N'-acyl-N,N'-dilyso-GM₁

N-n-octyl-sulfonyl-N'-acyl-N,N'-dilyso-GM₁

N-n-decyl-sulfonyl-N'-acyl-N,N'-dilyso-GM₁

N-2-bromoethyl-sulfonyl-N'-acyl-N,N'-dilyso-GM₁

N-hexadecyl-sulfonyl-N'-acyl-N,N'-dilyso-GM₁

N-3-chloropropyl-sulfonyl-N'-acyl-N,N'-dilyso-GM₁

N-6-bromohexyl-sulfonyl-N'-acyl-N,N'-dilyso-GM₁

N-benzyl-sulfonyl-N'-acyl-N,N'-dilyso-GM₁

N-4-chlorobenzyl-sulfonyl-N'-acyl-N,N'-dilyso-GM₁

N-4-aminobenzyl-sulfonyl-N'-acyl-N,N'-dilyso-GM₁

N-3,4,5-trimethoxybenzyl-sulfonyl-N'-acyl-N,N'-dilyso-GM₁

N-sulfo-N'-acyl-N,N'-dilyso-GM₁

and

N-acyl-N'-ethylsulfonyl-N,N'-dilyso-GM₁

N-acyl-N'-propyl-sulfonyl-N,N'-dilyso-GM₁

N-acyl-N'-n-butyl-sulfonyl-N,N'-dilyso-GM₁

N-acyl-N'-n-pentyl-sulfonyl-N,N'-dilyso-GM₁

N-acyl-N'-n-hexyl-sulfonyl-N,N'-dilyso-GM₁

N-acyl-N'-n-heptyl-sulfonyl-N,N'-dilyso-GM₁

N-acyl-N'-n-octyl-sulfonyl-N,N'-dilyso-GM₁

N-acyl-N'-n-decyl-sulfonyl-N,N'-dilyso-GM₁

N-acyl-N'-2-bromoethyl-sulfonyl-N,N'-dilyso-GM₁

N-acyl-N'-hexadecyl-sulfonyl-N,N'-dilyso-GM₁

N-acyl-N'-3-chloropropyl-sulfonyl-N,N'-dilyso-GM₁

N-acyl-N'-6-bromohexyl-sulfonyl-N,N'-dilyso-GM₁

N-acyl-N'-benzyl-sulfonyl-N,N'-dilyso-GM₁

N-acyl-N'-4-chlorobenzyl-sulfonyl-N,N'-dilyso-GM₁

N-acyl-N'-4-aminobenzyl-sulfonyl-N,N'-dilyso-GM₁

N-acyl-N'-3,4,5-trimethoxybenzyl-sulfonyl-N,N'-dilyso-GM₁

N-acyl-N'-sulfo-N,N'-dilyso-GM₁

wherein "acyl" designates the acyl radical of acetic acid, chloroaceticacid, dichloroacetic acid, propionic acid, n-valerianic acid,trimethylacetic acid, caproic and hexanoic acid, caprylic acid andundecylic acid and the esters thereof with alcohols selected from thegroup consisting of ethyl alcohol, propyl alcohol, n-butyl alcohol,n-pentyl alcohol, n-hexyl alcohol, n-heptyl alcohol, n-octyl alcohol,n-decyl alcohol, 2-bromoethyl alcohol, hexadecyl alcohol,3-chloropropylalcohol, 6-bromohexylalcohol, benzyl alcohol,4-chlorobenzyl alcohol, 4-aminobenzyl alcohol and 3,4,5-trimethoxybenzylalcohol.

With these derivatives it is also possible to prepare the correspondingesters of sulfuric acid at the hydroxyl groups, i.e. partially ortotally sulfated derivatives.

THERAPEUTIC ACTIVITY

It is well known that gangliosides are glycosphingolipids containingsialic acid with a basic saccharide structure bound to ceramide and oneor more molecules of sialic acid. The saccharide portion presents atleast one galactose or glucose unit and one N-acetylglucosamine orN-acetylgalactosamine.

The general structure of a ganglioside can so be represented as follows:##EQU2## where all the components are bound by glucosidic bonds.

A large number of gangliosides have been identified which areparticularly abundant in the nervous tissue, especially in the cerebralone (Ando S.: Gangliosides in the nervous system. Neurochem. Int. 5,507537, 1983).

It has been widely demonstrated that gangliosides are able to enhancefunctional recovery both in the lesioned Periferal Nervous System (PNS)and Central Nervous System (CNS), through the involvement of specificmembrane mechanisms and the interaction with trophic factors, as pointedout from studies in vitro on neuronal cultures (Ferrari F. et al.: Dev.Brain Res., 8:215-221, 1983; Doherty P. et al., J. Neurochem. 44:1259-1265, 1985; Skaper S. D. et al., Mol. Neurobiol. 3:173-199, 1989).

Moreover, it has been shown that gangliosides are able to selectivelyact where mechanisms responsible for neurotoxicity have been activated,thus antagonizing the effects of the paroxysmal and continuousstimulation of the excitatory amino acid receptors (Favaron M. et al.:Gangliosides prevent glutamate and kainate neurotoxicity in primaryneuronal cultures of neonatal rat cerebellum and cortex. Proc. Natl.Acad. Sci. 85: 7351-7355, 1988).

Concerning the PNS, the effects of the ganglioside mixture have beenreported in models of traumatic (Gorio A. et al., Brain Res. 197:236241, 1980), metabolic (Norido F. et al., Exp. Neurol. 83: 221-232,1984) and toxic (Di Gregorio F. et al., Cancer Chemother. Pharmacol. 26:3136, 1990) neuropathies. Concerning the CNS, the positive effects ofrecovery induced by the monosialoganglioside GM₁ have been widelydescribed in ischemia models (Karpiak S. E. et al., CRC Critical Rev. inNeurobiology, vol. 5. Issue 3, pp. 221-237, 1990), as well as intraumatic (Toffano G. et al., Brain Res. 296: 233-239, 1984) andneurotoxic (Schneider et al., Science 256: 843-846, 1992) lesions. Suchresults have led to the clinical application of GM₁ in conditions ofischemic brain injury (Argentino C. et al., Stroke 20: 1143-1149, 1989)and in conditions of traumatic injury of the spinal cord (Geisler F. H.,N. Engl. J. Med. 324: 1829-1838, 1991).

In addition, it has been recently shown that gangliosides are involvedin the modulation of the expression of the receptors named CD₄, whichare present on the membrane of some lymphocytes and furthermore it hasbeen shown that such a modulation is associated with an inhibition ofthe proliferation of the HIV virus (Offner H. et al.: Gangliosidesinduce selective modulation of CD₄ from helper T lymphocytes. J.Immunol. 139: 3295-3305, 1987; Grassi F. et al.: Chemical residues ofganglioside molecules involved in interactions with lymphocyte surfacetargets leading to CD₄ masking and inhibition of mitogenicproliferation. Eur. J. Immunol. 20: 145-150, 1990; Chieco-Bianchi et al.CD₄ modulation and inhibition of HIV-1 infectivity induced bymonosialoganglioside GM₁ in vitro. AIDS 3:501-507, 1989).

The molecule named CD₄ is a membrane glycoprotein of 55 KDa expressed bythymocytes, by a "subset" of T lymphocytes and, at a lower density, bymonocytes/macrophages. The molecule can be divided into three portions:one extracellular, that is divided into 4 domains, three of which have astructure that joins them to the superfamily of immunoglobulins, anintra-membrane portion of 21 amino acids (aa) , and an intracytosolicportion of 40 basic aa.

In T lymphocytes CD₄ has at least two functions. On one hand, itinteracts with a non-polymorphic region of class II HLA molecules, thusstabilizing the bond between the T cell and the cell which expresses theantigen (secondary role). On the other hand, recent evidence have shownhow the interaction of CD₄ with its own ligand induces the activation ofa cytoplasmic tyrosine kinase (named p56 lck) that is in contact withthe intracytosolic portion of CD₄. The activation of the tyrosine kinaseand the subsequent phosphorylation of different substrates, among themthe gamma chain of CD₃, has a facilitatory role in the signaltransduction following the interaction between the antigen receptor andthe antigen itself. Hence, CD₄ has an active role in the mechanisms thatregulate the activation of T lymphocytes.

In addition to these relevant functions for the physiology of T cells,CD₄ is also the receptor utilized by HIV viruses to enter the targetcells.

The CD₄₊ T lymphocytes play a major role in immune system functioning.In the majority of cases, following contact with the antigen the firstcells responsible for any adaptative response are the CD₄₊ T cells,which after the activation, may become in turn effectors of response.Alternatively, the activated CD₄₊ cells can help, by release ofcytokines, other cells (B cells, CD₈₊ T cells) to become effectors ofresponse. This is valid both for responses against foreign antigens(non-self) and for body antigens (self). Thus, CD₄₊ T cells areprimarily involved in several autoimmune diseases.

The possibility of modulating the CD₄₊ T cell function is relevant in awide range of human pathologies.

In different models, both in vitro and in vivo, it has been shown thatby blocking the CD₄ molecule with monoclonal antibodies, the function ofCD₄₊ T cells is inhibited. Such an inhibition leads in turn tounsuccessful proliferation, unsuccessful production of cytokines,unsuccessful production of antibodies, and suppression or lowering ofclinical expression of autoimmune symptoms in experimental models ofautoimmune pathology.

The pharmacological properties of the new sulfated derivatives,according to the present invention, can be emphasized by theexperimental studies performed on the following compounds:

N-4-CHLOROBENZENESULFONYL-LYSO GM₁ (Liga 123)

N-BENZENESULFONYL-LYSO GM₁ (Liga 125)

N'-O-SULFO-LYSO GM₁ (Liga 179)

Hereinafter will be described the experimental models and the resultsobtained with some representative compounds of the present invention.

1. Antineurotoxic effect of Liga 123 in vitro in cerebellar granulecells: protective effect against neurotoxicity induced by exogenousglutamate.

MATERIALS AND METHODS

Cell cultures

Primary cultures of cerebellar granule cells have been prepared from8-day-old Sprague-Dawley rats.

Neurons have been grown in 35 mm dishes for 11-13 days and kept in ahumidified environment (95% air and 5% CO2) at 37° C. Cultures (2.5×10⁶cells/dish) were mainly constituted of granular cells (>95%) with a lowpercentage (<5%) of glial cells (Gallo V. et al.: Selective release ofglutamate from cerebellar granule cells differentiating in culture.Proc. Natl. Acad. Sci. USA 79, 7919-7923, 1982). Glial proliferation wasprevented by cytosine arabinofuranoside.

The Liga 123 derivative has been solubilized in chloroform/methanol(2/1), then dried under a nitrogen flow and diluted at differentconcentrations in Locke's solution (154 mM NaCl, 5.6 mM KCl, 3.6 mMNaHCO₃, 2.3 mM CaCl₂, 1 mM MgCl₂, 5.6 mM glucose, 4.6 mM Hepes, pH 7.4).

Concentrations from 100 μM to 5 μM have been tested.

Description of the model of neurotoxicity induced by exogenousglutamate.: compound in a pretreatment paradigm.

The cell culture medium was aspirated from the dishes (and properlystored). The dishes were washed (3×2 ml) with Locke's solution, then thesolutions (1.5 ml) containing the compound to be tested were added, andincubated for 2 hrs in incubator at 37° C. (5% CO₂).

The treated cells were washed (3×2 ml) with Locke's solution+10% fetalcalf serum heat inactivated (without glutamic acid), then washed (3×2ml) in Locke's solution in the absence of Mg²⁺. Glutamate was added at100 μM (1.5 ml) in Locke's solution (-Mg²⁺) or Locke's solution alone(Mg²⁺) was added (controls). The incubation with glutamate or withLocke's (-Mg²⁺) was performed for 60 minutes at room temperature (27°C.). The glutamate was then removed, the dishes were washed with Locke'ssolution (+Mg²⁺) (2×2 ml), then incubated in the presence of the initialmedium (properly stored) for 24 hr at 37° C. in incubator (5% CO₂).

At the end of the incubation the cell viability measured by using theMTT colorimetric test was evaluated (Mosmann T., Rapid colorimetricassay for cellular growth and survival: application to proliferation andcytotoxicity assays. J. Immunol. Meth. 65, 55-63, 1983 and modifiedaccording to Skaper S. D. et al.: Death of cultured hippocampalpyramidal neurons induced by pathological activation ofN-methyl-Daspartate receptors is reduced by monosialogangliosides. J.Pharm. and Exp. Ther. 259, 1, 452-457, 1991). The data are expressed asED₅₀ (μM).

RESULTS

The obtained results (Table 1) show that the Liga 123 compound has amarked antineurotoxic activity (ED₅₀ =6 μM): the neuroprotective effectof Liga 123, at a concentration of 25 μM, is about 74%.

                  TABLE 1                                                         ______________________________________                                        Antineuronotoxic effect of Liga 123 in                                        cerebellar granule cells: protective effect on                                neurotoxicity induced by exogenous glutamate                                  Groups       (concentrations μM)                                                                     % survival (±SD)                                 ______________________________________                                        1)    control                 100                                             2)    glutamate               19 ± 1                                       3)    glutamate  25 μM     74 ± 5                                             + Liga 123                                                                               10 μM     63 ± 6                                                         5 μM     35 ± 3                                       ______________________________________                                    

2. Antineurotoxic effect of Liga 179 in vitro in cerebellar granulecells: compound in cotreatment paradigm with glutamic acid

MATERIALS AND METHODS

Cell cultures

Primary cultures of cerebellar granule cells have been preparedaccording to the method described in Materials and Methods of thepreceding experiment (1).

The Liga 179 derivative has been dissolved in sterile water at aconcentration of 50 mM. Thus, dilutions have been performed at differentconcentrations in Locke's solution (154 mM NaCl, 5.6 mM KCl, 3.6 mMNaHCO₃, 2.3 mM CaCl₂, 5.6 mM glucose, 4.6 mM Hepes, pH 7.4).

Concentrations from 100 μM to 5 μM have been tested.

Compound in cotreatment paradigm with glutamic acid

The cell culture medium was aspirated from the dishes (and properlystored). The dishes were washed (3×2 ml) with Locke's solution in theabsence of Mg²⁺. Then, 1.5 ml of Locke's solution (-Mg²⁺) with orwithout 100 μM glutamate and with or without the compound to be testedwere added. The incubation period lasted 30 minutes (37° C.). Glutamateand the compound to be tested were then removed. The dishes were washedwith Locke's solution in the presence of Mg²⁺ (2×2 ml) and thenincubated in the presence of the initial medium (properly stored) for 24hours at 37° C. in an incubator (5% CO₂).

At the end of the incubation cell viability measured by means of the MTTcolorimetric test was evaluated (Mosmann T.: Rapid colorimetric assayfor cellular growth and survival: application to proliferation andcytotoxicity assays. J Immunol. Meth. 65, 55-63, 1983 and modifiedaccording to Skaper S. D. et al.: Death of cultured hippocampalpyramidal neurons induced by pathological activation ofN-methyl-D-aspartate receptors is reduced by monosialogangliosides. J.Pharm. and Exp. Ther. 259,1, 452-457, 1991). Data were expressed as ED₅₀(μM).

RESULTS

The data obtained (Table 2) show that the Liga 179 compound has a markedantineurotoxic activity (ED₅₀ =15 μM), even when administeredsimultaneously with glutamate (co-treatment): the neuroprotective effectof Liga 179 is clear already at 10 μM and reaches its maximum effect at50 μM.

                  TABLE 2                                                         ______________________________________                                        Antineurotoxic effect of Liga 179 in                                          simultaneous treatment with exogenous glutamate in                            cerebellar granule cells (protective effect).                                                             % survival                                        Groups         (Concentration μM)                                                                      (average ± s.d.)                               ______________________________________                                        1)    control (-Mg2+)           100                                           2)    glutamate                  33 ± 1                                    3)    glutamate    100 μM    125 ± 5                                          + Liga 179                                                                                  50 μM    133 ± 9                                                        10 μM     52 ± 4                                    ______________________________________                                    

3. Neuritogenic activity of the compounds Liga 123 and Liga 125

MATERIALS AND METHODS

Cell Cultures

Mouse neuroblastoma cells C1300, Neuro-2A clone (obtained from AmericanCell Type Culture Collection--Bethesda, Md.) have been seeded at adensity of 10,000 cells/well (24-Falcon) in tissue culture mediumcontaining Dulbecco's modified Eagle medium (DMEM, Gibco), 10% fetalcalf serum heat inactivated (FCS, lot 7201 Seromed), penicillin (100units per ml, Irvine) and L-glutamine (2 mM, Sigma). Cells have beenincubated at 37° C. for 24 hr, then the medium was withdrawn andsubstituted with 350 μl of fresh culture medium with and without thecompounds to be tested.

Compounds under examination and their solubilization

The derivatives have been solubilized in chloroform/methanol (2/1) andthen dried under a flow of nitrogen.

For the different compounds, consecutive dilutions in tissue culturemedium (concentrations from 50 μM to 5 μM) were performed.

Parameters: neuritogenic activity (% of neurite-bearing cells under theoptical microscope).

Culture dishes incubated with the tested compounds were analyzed under aphase contrast microscope (250×): 9 optical fields were chosen withprefixed coordinates and photographed. Then, the total number of cellswere counted, as well as the number of neurite-bearing cells (length atleast double of the cell diameter) in blind on every picture. Thepercentage of neurite-bearing cells was determined following thecounting of at least 100 cells, and the data were expressed by therespective ED₅₀ (μM) (Facci L. et al.: Promotion of neuritogenesis inmouse neuroblastoma cells by exogenous ganglioside GM₁. J. Neurochem.229-305, 1984).

RESULTS

The results obtained (Table 3) show that the Liga 123 and 125derivatives promote neuritogenesis in vitro. In particular, in theexperimental conditions tested, it turns out that:

the neuritogenic effect is remarkable for the Liga 123 derivative: at adose of 25 μM, about 48% of the cells present long and ramifiedneurites.

the neuritogenic effect with Liga 126 is maximal at a dose of 50 μM (56%of neurite-bearing cells).

                  TABLE 3                                                         ______________________________________                                        Neuritogenic effect of Liga 123 and 125 in                                    neuroblastoma cells N2A                                                              Compounds                                                                             ED.sub.50 (μM)                                              ______________________________________                                               Liga 123                                                                              12 μM                                                              Liga 125                                                                              25 μM                                                       ______________________________________                                    

4. Effect of Liga 179 on the expression of the CD₄ molecule in Molt 3cells

Molt 3 cells (American Type Culture Collection--Rockville, Md., USA)have been utilized, human tumoral cell lines derived from an acutelymphoblastic leukemia and formed by T lymphocytes expressing CD₄ ontheir surface. Such a cell line was chosen due to the fact that itoverlaps, as regards the expression of the CD₄ molecule, human Tlymphocytes obtained from peripheral blood. 100% of Molt 3 cells expressCD₄, whereas only part of human T lymphocytes from peripheral bloodexpress CD₄ in a proportion varying from subject to subject. The Molt 3model has therefore the advantage of allowing a better experimentalreliability.

MATERIALS AND METHODS

Molt 3 cells (1×10⁶) have been incubated with different concentrationsof Liga 179 (from 1 μg/ml to 500 μg/ml) for 60 minutes at 37° C. inbuffered saline (PBS), with or without Fetal Calf Serum (FCS). Whenutilized, FCS was added at a concentration of 5 or 10 part/percentage(vol/vol). Following the incubation and the subsequent washing thepercentage of cells expressing CD₄ has been measured by flowcytofluorimetry using a monoclonal, fluoresceinated (mAb), specific forCD₄ (DAKO T4, Dakopatts, Glostrup, Denmark) and a cytofluorimeter (EPICSV, Coulter Electronics, Hialeah, Fla., USA).

In Table 4 are reported the data concerning the percentage of Molt 3cells that express CD₄, following the incubation with the compound underexamination at the different concentrations utilized.

                  TABLE 4                                                         ______________________________________                                        Effect of Liga 179 on the expression of CD4 in                                Molt 3 cells                                                                  % MOLT-3 cells expressing CD.sub.4 on the surface                                           FCS (%)                                                         Compound     μg/ml                                                                             0          5    10                                        ______________________________________                                        Liga 179      0     96.9       98.6 96.9                                      Liga 179      10     0         --   --                                        Liga 179      50     0         --   --                                        Liga 179     100     0         15.5 86.5                                      Liga 179     200    --          0   14.6                                      Liga 179     500    --          0    0                                        ______________________________________                                    

The results reported in Table 4 show how the modulating effect of Liga179 can be expressed in a dose/response curve, and how increasing dosesof serum require increasing concentrations of Liga 179.

It is important to point out that, at the highest concentration ofserum, the Liga 179 compound is able to totally inhibit the expressionof CD₄ .

CONCLUSIONS

The above-described results show a remarkably interestingpharmacological profile of the new compounds, which are object of thepresent invention. Special mention should be made of the antineurotoxiceffect on CNS cells, and the modulatory effect on the expression of theCD₄ molecule in the immune system cells.

In consideration of the antineurotoxic effect, the novel derivatives ofthe neuraminic acid may be successfully used in disorders associatedwith an excitatory activity of the excitatory amino acids. It has beendemonstrated that such amino acids, e.g., the glutamic or aspartic acid,besides their major role in different physiological processes, e.g.synaptogenesis and neuronal plasticity, are involved in the etiogenesisand/or evolution of different disorders with neuronal dysfunctionsand/or death. Even though neuronal damage may have different causes, theneuronal dysfunctions trigger a cascade of cellular events, such as theactivation of enzymatic reactions depending on Ca⁺² ions, the influenceof Ca⁺² ions, the activation of secondary messengers, which result inneuronal death. Hence, a further aspect of the invention is directed toa method for treating conditions related to neurotoxicity induced byexcitatory amino acids which comprises administration of a compoundaccording to the invention, together with a pharmaceutically acceptableexcipient, to a patient in need therefor. Damage to the CNS caused byexcitatory amino acids appears for instance in ischemia, epilepsy,trauma, compression, metabolic dysfunctions, aging, toxic-infectivedisorders, as well as in chronic neurodegenerative disorders, such asAlzheimer's disease or Huntington's chorea (Engelsen B., Acta Neurol.Scand. "Neurotransmitter glutamate: its clinical importance", 186, 4,337355; Olney J. W., Annu. Rev. Pharmacol. Toxicol., "Exctatotoxic aminoacids and neuropsychiatric disorders", 1990, 30, 47-71).

In addition, the novel compounds which are object of the presentinvention, in consideration of their neurite-promoting activity, may beused with advantage in the treatments aiming at nerve function recoveryin those pathological conditions associated with a neuronal damage, suchas peripheral neuropathies.

Moreover, the capacity of such compounds to modulate the expression ofthe CD₄ molecule on immune cell surface, may be of great relevance in awide range of human pathologies, e.g., those situations in which it isnecessary to prevent and/or treat infections in which CD₄ cells areinvolved (especially infections whose etiological agents aremicroorganisms belonging to the HIV virus family). Moreover, modulationof CD₄ is useful in systemic or organ-specific autoimmune diseases, suchas multiple sclerosis, rheumatoid arthritis, chronic polyarthritis,lupus erythematosus, juvenile-onset diabetes mellitus and also toprevent the phenomenon of organ transplant rejection as well asrejection by the transplanted material against the host, as in the caseof bone marrow transplant, and in all cases where the desired effect isto obtain tolerance towards "self" and "non-self" antigens.

The present invention includes also processes for the preparation of newcompounds. Such processes involve conventional and well-documentedapproaches for the conversion of an amino group into its acylatedderivative or for the selective hydrolysis of acyl derivatives of aminogroups, and optionally for the esterification of functional groups,specifically, the carboxyl groups, or for the esterification of hydroxylgroups. Thus, the process for the preparation of new compounds consistsin treating a N,N'-di-lyso-ganglioside or one of its N- or N'-acylderivatives with sulfuric acid, a hydrocarboxyl-sulfuric acid or ahydrocarbylsulfonic acid or their reactive derivative, if desired,acylating an amino group in position N- or N'- and, if desired,hydrolyzing in the obtained compounds one of the two acylated aminogroups thereby converting them into free amino groups and optionallyconverting free carboxyl groups or free hydroxyl groups into theirfunctional derivatives and optionally converting the obtained compoundsinto their metal salts or salts deriving from organic bases, or intotheir acid addition salts.

The process also involves those modifications in which the procedure isinterrupted at any phase and, if desired, the remaining steps areperformed or in which the procedure is started from an intermediate andthe remaining steps are performed or in which an "in situ" intermediateis formed.

The lyso-gangliosides may be prepared from gangliosides or fromN-lyso-gangliosides by alkaline hydrolysis, for example withtetraalkylammonium hydroxides, sodium hydroxide or similar agents.

The preparation of N- or N'-mono or poly-acyl-derivatives fromN,N'-dilyso-gangliosides is described in literature.

Compounds having an acyl group on the neuraminic nitrogen can beprepared by various methods. It is possible, for example, to start withdilyso-gangliosides and then effect an intermediate provisionalprotection of the sphingosine amino group, which can be done for exampleby hydrophobic interaction with phosphatidylcholine, or by acylationwith suitable protective groups, subsequent acylation on the neuraminicnitrogen with a derivative of the acid which is to be introduced intothis position, and then deprotection on the sphingosine nitrogen.Alternatively, dilyso-gangliosides can be acylated on the two aminogroups with the same acid and the diacyl compound can be exposed to theaction of enzymes which are able to selectively remove the acylaminogroups from the sphingosine nitrogen, for example enzymes used to obtainlyso-gangliosides from gangliosides, such as theglycosphingolipid-ceramide-deacylase enzyme (see scheme 1).N-monoacyl-N,N'-dilyso-gangliosides can however also be obtained bydeacylating N,N'-diacyl-N,N'-dilyso-gangliosides on the neuraminicnitrogen by selective chemical hydrolysis, for example with 0.1 molaralcoholic potassium hydroxide.

The procedure for the preparation of N-acyl-N,N'-dilyso-gangliosidescomprises acylating N,N'-di-lyso-gangliosides with the acidscorresponding to the acyl group to be introduced or selectivelydeacylating suitable N,N'-diacyl-N,N'-dilyso-gangliosides on theneuraminic nitrogen.

N-acylation according to the aforesaid procedure can be effected in theconventional manner, for example by reacting the starting products withan acylating agent, especially with a functional derivative of the acid,the residue of which is to be introduced. Thus, it is possible to use ahalide or an anhydride as the functional derivative of the acid, and theacylation is carried out preferably in the presence of a tertiary base,such as pyridine or collidine. Anhydrous conditions can be used at roomtemperature or at higher temperatures, or the Schotten-Baumann methodcan also be used to advantage, operating in aqueous conditions in thepresence of an organic base. In some cases it is also possible to useesters of the acids as reactive functional derivatives. Of all thepreparation methods, the following are the most appropriate:

1. reaction of the lyso-ganglioside derivative with the azide of theacid;

2. reaction of the lyso-ganglioside derivative with an acylimidazole ofthe acid obtainable from the acid with N,N'-carbonyldiimidazole;

3. reaction of the lyso-ganglioside derivative with a mixed anhydride ofthe acid and of trifluoroacetic acid;

4. reaction of the lyso-ganglioside derivative with the chloride of theacid;

5. reaction of the lyso-ganglioside derivative with the acid in thepresence of a carbodiimid (such as dicyclohexylcarbodiimide) andoptionally a substance such as 1-hydroxybenzotriazole;

6. reaction of the lyso-ganglioside derivative with the acid by heating;

7. reaction of the lyso-ganglioside derivative with a methyl ester ofthe acid at a high temperature;

8. reaction of the lyso-ganglioside derivative with a phenol ester ofthe acid, for example an ester with para-nitrophenol;

9. reaction of the lyso-ganglioside derivative with an ester derivedfrom the exchange between a salt of the acid and1-methyl-2-chloropyridinium iodide.

It has already been explained how it is possible to obtain selectivepartial acylation both on the sphingosine and on the neuraminicnitrogen. Scheme 1 illustrates these procedures.

Enzymatic deacylation of N,N'-diacyl-N,N'- dilyso-gangliosides on thesphingosine nitrogen as previously reported can be effected under thesame conditions as those used for the partial deacylation ofgangliosides, for example as described in J. Biochem., 103, 1 (1988).The double deacylation of N,N'-diacyl-N,N'-dilyso-gangliosides toN,N'-dilyso-gangliosides can be effected in the same way as thepreparation of de-N-acetyl-lyso-gangliosides as described for example inBiochemistry 24, 525 (1985); J. Biol. Chem. 255, 7657, (1980); Biol.Chem. Hoppe Seyler 367, 241, (1986): Carbohydr. Research 179, 393(1988); Bioch. Bioph. Res. Comn. 147, 127 (1987).

The aforesaid publication in Carbohydr. Research 179 also describes amethod for selective deacylation on the neuraminic nitrogen by theaction of KOH (0.1M) in 90% normal butanol with the ganglioside GM₃.This type of deacylation reaction can be applied toN,N'-diacyl-N,N'-dilyso-gangliosides to obtainN-acyl-N,N'-dilyso-gangliosides. ##STR4##

The conversion of N,N'-dilyso-gangliosides into the aforesaid di- andpolyderivatives of sulfuric acid, of its esters or ofhydrocarbylsulfonic acids may be performed partially, the sphingosineamino group being more reactive than the neuraminic amino group. It istherefore possible to obtain the N-derivatives with the amino groupconverted with the corresponding acid, and it is also possible, if onewishes, to convert in the same manner the neuraminic amino group in asecond step.

The insertion of the sulfo (SO₂ OH) group into an amino group of theaforesaid N,N'-dilyso-gangliosides (or in both, in all the amino groupsrespectively) is preferably done with the reactive derivative of thesulfuric acid and its trioxide, respectively; normally, the sulfurtrioxide-pyridine complex is used, provided an adequate solvent, forexample water, at room temperature.

The insertion of a hydrocarbyl-sulfonyl group is preferably done with ahalide such as chloride of the hydrocarbon-sulfonic acid, for examplewith the chloride or bromide of methanesulfonic or p-toluene-sulfonicacid, in the presence of a tertiary base, such as pyridine, collidine,triethylamine, etc.

The insertion of a hydrocarbyloxy-sulfonyl group occurs by reaction withthe corresponding ether of the chlorosulfonic acid, also in the presenceof a tertiary base, such as one of the above-mentioned ones.

The optional acylation of the amino groups in position N- or N'- in thecompounds containing only one of the said sulfured groups is preferablydone with an acylating agent, preferably with a reactive functionalderivative of the acid from which the acyl group is to be inserted.Therefore it is possible to use a halide or an anhydride as a functionalderivative, and the acylation is preferably done in the presence of atertiary base, such as pyridine or collidine. It is also possible towork in an anhydrous environment, at room temperature or heating; oreven successfully following the method of Schotten-Baumann in an aqueousenvironment, in the presence of an inorganic base.

For the acylation it is also possible to use methods involving activatedcarboxyl derivatives, such as the ones used in the chemistry ofpeptides, for example the method of the mixed anhydrides or derivativesobtainable with carbodiimide derivatives or the isoxazole salts.

The functional modification to be eventually performed, if desired, onthe compounds obtained from the conversion with the said derivatives ofsulfured acids or with organic acids is also done according to thewell-known methods, hence excluding those methods which could affect thebasic ganglioside structure, such as those involving highly acidicagents, or which anyway would be performed in critical alkaline oracidic conditions, or also those methods that would bring forth anunwanted alkylation of the hydroxyl groups of the saccharide, sialic orceramide portion.

The esterification of the sialic carboxyl groups or their conversioninto amides can be performed for example as described in the U.S. Pat.No. 4,713,374 of Dec. 12, 1987.

Amides can be prepared for example according to the following methods:

a) reaction of the carboxyl esters with ammonia or with amines;

b) reaction of derivatives according to the invention with carboxylgroups activated with ammonia or with the amines.

Acylation of the hydroxyl groups on the saccharide, sialic and ceramideportion can be performed, for example by means of an acyl halide or anacid anhydride, preferably in the presence of a tertiary base.

The conversion of the hydroxyl groups into the esters of sulfuric acid,which is to be emphasized in the present invention, can be performed ina well-known manner, for example and, preferably, with the sulfurtrioxide/dimethylformamide complex in the presence of a base, such astriethylamine, or with the sulfur trioxide/trimethylamine indimethylformamide complex, then with trifluoroacetic acid indichloromethane. (compare: Biochemical and Biophysical ResearchCommunications, Vol. 175, No. 1, Feb. 28, 1991).

With such methods, which can vary according to temperature conditions,solvents used and duration of the reaction, it is possible to obtainpartial sulfuric esters of the hydroxyl groups or total esters, hencepersulfated compounds.

Referred to the present invention are also pharmaceutical preparationsincluding, as active ingredients, one or more of the novel compoundsand, in particular, those above emphasized. Such preparations can beassigned to the oral, rectal, parenteral, local or transdermicadministration, thus being in a solid or semisolid form, for examplepills, tablets, gelatine capsules, capsules, suppositories, softgelatine capsules. For parenteral use, predetermined forms forintramuscular or transdermic administration, or forms suitable forinfusions or intravenous injection are considered, and they can betherefore prepared as solutions of the active ingredients, or aslyophilized forms of the active ingredients to be mixed before use withone or more excipients or pharmaceutically acceptable solvents, suitableto these uses and osmolarity-compatible with the physiological fluids.

For local administration preparations in the form of sprays, for examplenasal sprays, creams and ointments for topical use or bandage adequatelyprepared for transdermic administration are considered.

The pharmaceutical preparations of the present invention can beadministered both to humans and animals in need therefor. They containpreferably 0.01% to 0.1% of the active ingredient in the case ofsolutions, sprays, ointments and creams, and 1% to 100%--preferably 5%to 50%--of the active ingredient for in the case of solid preparations.Dosage depends oil tile indication, the desired effect, and preferredroute of administration, as well as the weight and condition of thepatient treated.

The present invention also concerns the therapeutic use of the newsemisynthetic analogues for the above-said indications. The daily doseto be administered in human by parenteral route (subcutaneous orintramuscular), or by transdermic or oral route, is generally between0.5 and 5 mg of the active ingredient per kg of body weight. In thepreparations reported hereinafter a dose of 150 mg per unit can bereached.

The following examples illustrate the preparation of the newsemisynthetic analogues which are object of the present invention, aswell as the preparations that contain them as active ingredients.

EXAMPLE 1 N-SULFO-LYSO GM₁

500 mg (0.39 mM) of N-deacyl-lyso GM₁ (N-lyso GM₁) are dissolved in 50ml of Na₂ CO₃ 0.2M in water. 248.3 ml (1.56 mM) of a sulfurtrioxide-pyridine complex are then added; the reaction lasts 15 min atroom temperature.

Once the reaction is achieved, dialyze in water, concentrate to 100mg/ml and precipitate in 10 volumes acetone.

Obtained compound: 470 mg (87% theoretical). The name of the compound isalso N-sulfo-N'-acetyl-N,N'-dilyso GM₁.

Chromatographed on silica gel plate with chloroform/methanol/CaCl₂ 0.3%60/35/8, it proves to be a unitary compound with Rf=0.14 (GM₁ =0.40,N-deacyl-lyso GM₁ =0.17).

EXAMPLE 2 N-SULFO-LYSO GM₂

100 mg (0.071 mM) of GM₂ are dissolved in 2 ml KOH 3M and reaction lasts60 hours at 90° C. Once the reaction is achieved, cool the solution andbring it to pH 6.5 with hydrochloric acid. Keep for 18 hours at 4° C.,then filtrate the separated fatty acids. Dialyze against H₂ O,concentrate and precipitate in 5 volumes of acetone.

The obtained compound, deacyl-deacetyl-GM₂ (N,N'-dilyso GM₂), ishigh-vacuum dried and again dissolved in 1 ml of dimethylformamide.

30 mg of 9-fluorenylmethyloxycarbonyl-N-hydroxysuccinimide dissolved in0.5 ml tetrahydrofurane are slowly added, and the reaction lasts 1 hourat room temperature. Once the reaction is achieved, add 50 μl of aceticanhydride and react for 30 min. Supplement with 150 μl of pyridine inorder to remove the protective compound, react for 18 hours at roomtemperature, precipitate in 10 volumes of acetone and dry.

The obtained compound is then dissolved in Na₂ CO₃ 1M and kept for 1hour at 60° C. Dialyze, concentrate to 0.5 ml and precipitate in 5volumes of acetone.

The compound is then passed a through S-Sepharose column (H+ form)balanced in methanol. Wash with methanol and dissolve N-lyso-GM₂ withNH₄ Cl 10 nM in methanol. The compound is dried, again water-dissolved,brought to pH 10 with NaOH 0.01N, again dialyzed and then lyophilized.

20 mg of sulfur trioxide-pyridine complex are then added and thereaction lasts 15 min at room temperature. Once the reaction isachieved, dialyze in water, concentrate at 0.5 ml and precipitate in 10volumes of acetone.

Obtained compound: 48 mg

Chromatographed on silica-gel plate with chloroform/methanol/CaCl₂ 0.3%60/35/8, it proves to be a unitary compound with Rf=0.18 (GM₂ =0.52).

EXAMPLE 3 N-SULFO-LYSO GM₃

100 mg (0.084 mM) of GM₃ are dissolved in 2 ml KOH 3M and the reactionlasts 60 hours at 90° C. Once the reaction is achieved, cool and bringto pH 6.5 with hydrochloric acid. Keep for 18 hours at 4° C. and thenfiltrate the separate fatty acids. Dialyze against H₂ O, concentrate andprecipitate in 5 volumes of acetone.

The compound obtained, deacyl-deacetyl-GM₃ (N,N'-dilyso GM₃), ishigh-vacuum dried and again dissolved in 1 ml of dimethylformamide.

30 mg of 9-fluorenylmethyloxycarbonyl-N-hydroxysuccinimide dissolved in0.5 ml tetrahydrofurane are slowly added and reacted for 1 hour at roomtemperature. At the end, supplement with 50 μl of acetic anhydride andreact for 30 min. Then 150 μl of piperidine are added in order to removethe protective compound; react for 18 hours at room temperature,precipitate in 10 volumes of acetone and dry.

The obtained compound is then dissolved in Na₂ CO₃ 1M and kept at 60° C.for 1 hour. Dialyze, concentrate to 0.5 ml and precipitate in 5 volumesof acetone.

The compound is then passed through a S-Sepharose column (H+ form)balanced in methanol. Wash with methanol and then dissolve deacyl-GM₃(N-lyso GM₃) with NH₄ Cl 10 nM in methanol. The compound is dried, againwater-dissolved, brought to pH 10 with NaOH 0.01N, again dialyzed andthen lyophilized.

Add 20 mg of sulfur trioxide-pyridine complex and react for 15 min atroom temperature. Once the reaction is achieved, dialyze against H₂ O,concentrate at 0.5 ml and precipitate in 10 volumes of acetone.

Obtained compound: 45 mg

Chromatographed on silica-gel plate with chloroform/methanol/CaCl₂ 0.3%60/35/8, it proves to be a unitary compound with Rf=0.23 (GM₃ =0.67).

EXAMPLE 4 N'-SULFO-GM₁

500 mg (0.33 mM) of deacetyl GM₁ (N'-lyso GM₁) are dissolved in 50 mlNa₂ CO₃ 0.2M in H₂ O. 210.1 mg (1.32 mM) of sulfur trioxide/pyridinecomplex are added and reacted for 15 min at room temperature. Once thereaction is achieved, dialyze against H₂ O, concentrate at 100 mg/ml andprecipitate in 10 volumes of acetone.

Obtained compound: 460 mg (86% theoretical)

Chromatographed on silica-gel plate with chloroform/methanol,/CaCl₂ 0.3%60/35/8, it proves to be a unitary compound with Rf=0.18 (GM₁ =0.40,N'-deacetyl GM₁ =0.20).

EXAMPLE 5 N-SULFO-LYSO GD_(1a)

500 mg (0.26 mM) of GD_(1a) are dissolved in 10 ml KOH 3M and thereaction lasts 60 hours at 90° C. Once the reaction is achieved, cooland bring to pH 6.5 with hydrochloric acid. Keep for 18 hours at 4° C.and then filtrate the separated fatty acids. Dialyze against H₂ O,concentrate and precipitate in 5 volumes of acetone.

The compound obtained, deacyl-deacetyl-GD_(1a) (N,N'-dilyso GD_(1a)), ishigh-vacuum dried and again dissolved in 5 ml of dimethylformamide.

150 mg of 9-fluorenylmethyloxycarbonyl-N-hydroxysuccinimide dissolved in2.5 ml tetrahydrofurane are slowly added and reacted for 1 hour at roomtemperature. At the end, supplement with 0.5 ml of acetic anhydride andreact for 30 min. One ml of piperidine is then added in order to removethe protective compound, react for 18 hours at room temperature,precipitate in 10 volumes of acetone and dry.

The obtained compound is then dissolved in Na₂ CO₃ 1M and kept at 60° C.for 1 hour. Dialyze, concentrate at 2.5 ml and precipitate in 5 volumesof acetone.

The compound is passed through a S-Sepharose column (H+ form) balancedin methanol. Wash with methanol and then dissolve deacylGD_(1a) with NH₄Cl 10 nM in methanol. The compound is dried, again water-dissolved,brought to pH 10 with NaOH 0.01N, again dialyzed and then lyophilized.

20 mg of the sulfur trioxide/pyridine complex are added and reacted for15 min at room temperature. Once the reaction is achieved, dialyzeagainst H₂ O, concentrate at 0.5 ml and precipitate in 10 volumes ofacetone.

Obtained compound: 225 mg

Chromatographed on silica-gel plate with chloroform/methanol/CaCl₂ 0.3%60/35/8, it proves to be a unitary compound with Rf=0.12 (GD_(1a)=0.37).

EXAMPLE 6 N,N'-DISULFO-N,N'-DILYSO GM₁

250 mg (0.20 mM) of N,N'-dilyso GM₁ are dissolved in 5 ml Na₂ CO₃ 0.2M,then add 300 mg of the sulfur trioxide/pyridine complex and react atroom temperature.

At the end, dialyze against H₂ O, concentrate at 100 mg and precipitatein 10 volumes of acetone.

Chromatograph on silica-gel plate with chloroform/methanol/H₂ O 60/30/6,collect pure fractions, concentrate and again precipitate in acetone.

Obtained compound: 110 mg

Chromatographed on silica-gel plate with chloroform/methanol/CaCl₂ 0.3%60/35/8, it proves to be a unitary compound with Rf=0.13(N,N'-dilyso-GM₁ =0.29).

EXAMPLE 7 N'-SULFO-GM₃

100 mg (0.084 mM) of GM₃ are dissolved in 10 ml NaOH 1N and react for 15hours at 90° C. Once the reaction is achieved, neutralize with HCl 1Nand partition with 5 volumes of chloroform/methanol 2/1.

Dry the organic phase with 15 ml of Na₂ CO₃ 0.2 M and react with 200 mgof sulfur trioxide/pyridine complex (sulfonating agent/gangliosideratio: 2/1) for 3 hours at room temperature.

Concentrate and dialyze against distilled H₂ O. Dry and chromatograph onsilica-gel plate with chloroform/methanol/NH₃ 5N 60/25/4.

Obtained compound: 70 mg

Chromatographed on silica-gel plate with chloroform/methanol/NH₃ 5N60/35/8, it proves to be a unitary compound with Rf=0.56 (GM₃ 0.67,deacetyl-GM₃ =0.48).

EXAMPLE 8 N-ACETYL,N'-SULFO-DILYSO GM₁

500 mg (0.37 mM) of N-acetyl-N,N'-dilyso GM₁ are dissolved in 20 ml ofNa₂ CO₃ 0.2 M; react with 250 mg of sulfur trioxide/pyridine complex(sulfonating agent/ganglioside ratio: 20/1) for 3 hours at roomtemperature.

Concentrate and dialyze against distilled H₂ O. Dry and chromatograph onsilica-gel plate with chloroform/methanol/NH₃ 5N 60/35/8.

Obtained compound: 325 mg

Chromatographed on silica-gel plate with chloroform/methanol/NH₃ 5N60/35/8., it proves to be a unitary compound with Rf=0.58(de-N'acetyl,N-acetyl-dilyso GM₁ =0.28).

EXAMPLE 9 N-4-CHLOROBENZENESULFONYL-LYSO GM₁

500 mg (0.39 mM) of N-lyso GM₁ are dissolved in 25 ml dimethylformamide.Supplement with 2 ml (14.4 mM) of triethylamine and then 1.6 g (7.58 mM)of 4-chloro benzenesulfonyl chloride.

React at room temperature for 24 hours. Add 1 g sodium acetate and dryby means of a rotating evaporator, re-suspend with sodium carbonate 1Nand saponify for 18 hours at 70° C.

The resulting raw compound is dialyzed against H₂ O, dried and purifiedby means of silica gel chromatography with chloroform/methanol/ammoniumcarbonate 3.2% in water, 60/25/4.

Collect pure fractions, dry, re-suspend with sodium carbonate 1N,dialyze against distilled H₂ O and then precipitate in 100 ml acetone.

Obtained compound: 120 mg

Chromatographed on silica-gel plate with chloroform/methanol/CaCl₂ 0.3%60/35/8, it proves to be a unitary compound with Rf=0.30 (GM₁ =0.40,N-deacyl-lyso GM₁ =0.17).

EXAMPLE 10 N-BENZENESULFONYL-LYSO GM₁

500 mg (0.39 mM) of N-lyso GM₁ are dissolved in 10 ml dimethylformamide.Take solution to 0° C. Supplement with 0.118 ml (0.84 mM) oftriethylamine and then 0.106 g (0.83 mM) of benzenesulfonylchloride.

React at room temperature for 24 hours at 0° C.

Add 1 g sodium acetate and dry by means of a rotating evaporator.

The resulting raw compound is dialyzed against H₂ O, dried and purifiedby means of silica gel chromatography with chloroform/methanol/ammoniumcarbonate 3.2% in water 60/25/4.

Pure fractions are collected, dried, re-suspended with sodium carbonate1N, dialyzed against distilled H₂ O and then precipitated in 100 mlacetone.

Obtained compound: 150 mg

Chromatographed on silica-gel plate with chloroform/methanol/CaCl₂ 0.3%60/35/8, it proves to be a unitary compound with Rf=0.30 (GM₁ =0.40,N-deacyl-lyso GM₁ =0.17).

EXAMPLE 11 N-METHANESULFONYL-LYSO GM₁

500 mg (0.39 mM) of N-lyso GM₁ are dissolved in 10 ml dimethylformamide.Supplement with 112 ml (0.76 mM) of methansulfonylimidazole.

React at room temperature for 72 hours. Add 1 g sodium acetate and dryby means of a rotating evaporator, resuspend with sodium carbonate 1Nand saponify for 18 hours at 70° C.

The resulting raw compound is dialyzed against H₂ O, dried and purifiedby means of silica gel chromatography with chloroform/methanol/ammoniumcarbonate 3.2% in water 60/25/4.

Pure fractions are collected, dried, re-suspended with sodium carbonate1N, dialyzed against distilled H₂ O and then precipitated in 100 mlacetone.

Obtained compound: 100 mg

Chromatographed on silica-gel plate with chloroform/methanol/CaCl₂ 0.3%60/35/8, it proves to be a unitary compound with Rf=0.20 (GM₁ =0.40,N-deacyl-lyso GM₁ =0.17).

EXAMPLE 12 N-2-BROMOETHANESULFONYL-LYSO GM₁

500 mg (0.39 mM) of N-deacyl-lyso GM₁ are dissolved in 10 mldimethylformamide. Supplement with 316 μl (2.2 mM) of triethylamine, 160mg (0.76 mM) of the 2-bromoethanesulfonic acid sodium salt and 194 mg(0.76 mM) of chloromethylpyridine iodide.

React at room temperature for 72 hours.

Add 1 g sodium acetate and dry the compound by means of a rotatingevaporator, re-suspend with sodium carbonate 1N and saponify for 18hours at 70° C.

The resulting raw compound is dialyzed against H₂ O, dried and purifiedby means of silica gel chromatography with chloroform/methanol/ammoniumcarbonate 3.2% in water 60/25/4.

Pure fractions are collected, dried, re-suspended with sodium carbonate1N, dialyzed against distilled H₂ O and then precipitated in 100 mlacetone.

Obtained compound: 126 mg

Chromatographed on silica-gel plate with chloroform/methanol/CaCl₂ 0.3%60/35/8, it proves to be a unitary compound with Rf=0.22 (GM₁ =0.40,N-deacyl-lyso GM₁ =0.17, N'-deacetyl-lyso GM₁ 0.20).

EXAMPLE 13 N-HEXADECANESULFONYL-LYSO GM₁

500 mg (0.38 mM) of N-lyso GM₁ are dissolved in 2.5 mldimethylformamide. Add at room temperature 316 μl (2.28 mM) oftriethylamine, 7 mg (0.83 mM) of hexadecanesulfonic acid sodium salt and194.2 mg (0.76 mM) of chloromethylpyridine iodide dissolved in 2.5 mldimethylformamide.

React at room temperature for 18 hours.

Precipitate in 100 ml acetone. Filtrate and dry. Purify by means ofsilica gel chromatography with chloroform/methanol/H₂ O 60/30/6.

Pure fractions are collected, dried, re-suspended with Na₂ C₃ 1N,dialyzed against distilled H₂ O, concentrated at 5 ml and precipitatedin 100 ml acetone.

Obtained compound: 2,05 mg

Chromatographed on silica-gel plate with chloroform/methanol/CaCl₂ 0.3%60/35/8, it proves to be a unitary compound with Rf=0.38 (GM₁ =0.40,N-deacyl-lyso GM₁ =0.17).

EXAMPLE 14 N-PYRIDINESULFONYL-LYSO GM₁

500 mg (0.38 mM) of N-lyso GM₁ are dissolved in 2.5 mldimethylformamide. Add at room temperature 316 μl (2.28 mM) oftriethylamine, 132 mg (0.83 mM) of pyridinesulfonic acid and 194.2 mg(0.76 mM) of chloromethylpyridine iodide dissolved in 2.5 mldimethylformamide.

React at room temperature for 18 hours.

Precipitate in 100 ml acetone. Filtrate and dry. Purify by means ofsilica gel chromatography with chloroform/methanol/H₂ O 60/30/6.

Pure fractions are collected, dried, re-suspended with Na₂ CO₃ 1N,dialyzed against distilled H₂ O, then concentrated at 5 ml andprecipitated in 50 ml acetone.

Obtained compound: 240 mg

Chromatographed on silica-gel plate with chloroform/methanol/CaCl₂ 0.3%60/35/8, it proves to be a unitary compound with Rf=0.35 (GM₁ =0.43,N-deacyl-lyso GM₁ =0.24) and plate-fluorescent at 254 nm.

EXAMPLE 15 N-O-SULFO-LYSO GM₁

500 mg (0.36 mM) of N-sulfo-lyso GM₁ are dissolved in 5 mldimethylformamide. Add at room temperature 0.5 ml (3.6 mM) oftriethylamine, and 276 mg (1.8 mM) of the sulfurtrioxide/dimethylformamide complex.

React under stirring at room temperature for 5 hours. Precipitate in 10ml acetone. Dissolve the resulting raw compound in 50 ml Na₂ CO₃ 1%,dialyze against H₂ O and lyophilize.

Obtained compound: 650 mg

Chromatographed on silica-gel plate with chloroform/methanol/CaCl₂ 0.3%60/35/8, it shows a Rf ranging from 0.09 to 0.15. Molar ratio of sulfategroups/neuraminic acid 5/1 (determination of sulfate groups by means ofion chromatography and determination of the neuraminic acid by means ofthe resorcinol method). Specific absorption I.R. S═O groups: 120 cm⁻¹(KRb).

EXAMPLE 16 N'-O-SULFO-LYSO GM₁

500 mg (0.31 nM) of N'-sulfo-lyso GM₁ are dissolved in 5 ml anhydrousdimethylformamide. Add at room temperature 0.43 ml (3.1 mM) oftriethylamine, and 237 mg (1.55 mM) of the sulfurtrioxide/dimethylformamide complex.

React under stirring at room temperature for 5 hours and precipitate in5 volumes of acetone.

Dissolve the resulting raw compound in 50 ml Na₂ CO₃ 1%, dialyze againstH₂ O and lyophilize.

Obtained compound: 620 mg

Chromatographed on silica-gel plate with chloroform/methanol/CaCl₂ 0.3%60/35/8, it shows a Rf ranging from 0.01 to 0.05. Molar ratio of thesulfate groups/neuraminic acid 5/1 (determination of sulfate groups bymeans of ion chromatography and determination of the neuraminic acidusing the resorcinol method). Specific absorption I.R. S═O groups: 1260cm⁻¹ (KRb).

EXAMPLE 17 INJECTABLE PHARMACEUTICAL PREPARATIONS

Preparation No. 1

One 2-ml vial contains:

Active ingredient 5 mg

Sodium chloride 16 mg

Citrate buffer pH=6

In water for injection, q.s. to 2 ml

Preparation No. 2

One 2-ml vial contains:

Active ingredient 50 mg

Sodium chloride 16 mg

Citrate buffer pH=6

In water for injection, q.s. to 2 ml

Preparation No. 3

One 4-ml vial contains:

Active ingredient 100 mg

Sodium chloride 32 mg

Citrate buffer pH =6

In water for injection, q.s. to 4 ml

EXAMPLE 18 PHARMACEUTICAL PREPARATIONS IN 2 VIALS

These preparations are prepared with 2 vials. The first vial containsthe active ingredient in the form of a lyophilized powder in quantitiesvarying from 10% to 90% in weight, together with a pharmaceuticallyacceptable excipient, such a glycine or mannitol. The second vialcontains a solvent, such as sodium chloride and a citrate buffer.

The contents of both ampules are mixed up immediately beforeadministration and the lyophilized active ingredient is rapidlydissolved, thus resulting in an injectable solution.

Method No. 1

a. One 2-ml vial of lyophilized powder contains:

Active ingredient 5 mg

Glycine 30 mg

b. One 2-ml vial of solvent contains:

Sodium chloride 16 mg

Citrate buffer 2 mg

In water for injection, q.s. to 2 ml

Method No. 2

a. One 3-ml vial of lyophilized powder contains:

Active ingredient 5 mg

Mannitol 40 mg

b. One 2-ml vial of solvent contains:

Sodium chloride 16 mg

Citrate buffer

In water for injection, q.s. to 2 ml

Method No. 3

a. One 3-ml vial of lyophilized powder contains:

Active ingredient 50 mg

Glycine 25 mg

b. One 3-ml vial of solvent contains:

Sodium chloride 24 mg

Citrate buffer

In water for injection, q.s. to 3 ml

Method No. 4

a. One 3-ml vial of lyophilized powder contains:

Active ingredient 50 mg

Mannitol 20 mg

b. One 3-ml vial of solvent contains:

Sodium chloride 24 mg

Citrate buffer

In water for injection, q.s. to 3 ml

Method No. 5

a. One 5-ml vial of lyophilized powder contains:

Active ingredient 150 mg

Glycine 50 mg

b. One 4-ml vial of solvent contains:

Sodium chloride 32 mg

Citrate buffer

In water for injection, q.s. to 4 ml

Method No. 6

a. One 5-ml vial of lyophilized powder contains:

Active ingredient 100 mg

Mannitol 40 mg

b. One 4-mi vial of solvent contains:

Sodium chloride 32 mg

Citrate buffer

In water for injection, q.s. to 4 ml

Method No. 7

a. One 3-ml vial of lyophilized powder contains:

Active ingredient

micronized, sterile 40 mg

b. One 3-ml vial of solvent contains:

Tween 80® 10 mg

Sodium chloride 24 mg

Phosphate buffer

In water for injection, q.s. to 3 ml

Method No. 8

a. One 5-ml vial of lyophilized powder contains:

Active ingredient

Micronized, sterile 100 mg

b. One 4-ml vial of solvent contains:

Tween 80® 15 mg

Soybean lecithin 5 mg

Sodium chloride 36 mg

Citrate buffer

In water for injection, q.s. to 4 ml

EXAMPLE 19 PHARMACEUTICAL PREPARATIONS FOR TRANSDERMAL ADMINISTRATION

Preparation No. 1.

A bandage contains:

Active ingredient 100 mg

Glycerol 1.6 g

Polyvinyl alcohol 200 mg

Polyvinyl pyrrolidone 100 mg

Excipient to increase

Transdermal penetration 20 mg

Water 1.5 g

Preparation No. 2.

100 g ointment contain:

Active ingredient 4.0 g

(in 5 g phospholipid liposomes)

Polyethylene glycol monostearate 1.5 g

Glycerol 1.5 g

Beta-oxybenzoic acid ester 125 mg

Water 72.9 g

EXAMPLE 20 PHARMACEUTICAL PREPARATIONS FOR ORAL ADMINISTRATION

Preparation No. 1.

A tablet contains:

Active ingredient 20 mg

Single-crystal cellulose 150 mg

Lactose 20 mg

Starch 10 mg

Magnesium stearate 5 mg

Preparation No. 2.

A pill contains:

Active ingredient 30 mg

Carboxymethyl cellulose 150 mg

Starch 15 mg

Lactose 10 mg

Sucrose 35 mg

Coloring agent 0.5 mg

Preparation No. 3.

A gelatine capsule contains:

Active ingredient 40 mg

Lactose 100 mg

Gastroresistant covering 5 mg

Preparation No. 4.

A soft gelatine capsule contains:

Active ingredient 50 mg

Vegetable oil 200 mg

Beeswax 20 mg

Gelatine 150 mg

Glycerol 50 mg

Coloring agent 3 mg

This invention being thus described, it is obvious that these methodscan be modified in various ways. Such modifications are not to beconsidered as divergences from the very spirit and purpose of theinvention, and any modification that would appear evident to an expertin the field comes within the scope of the following claims:

We claim:
 1. Semisynthetic ganglioside analogues, having a sialic acidresidue portion and oligosaccharide hydroxyl groups, selected from thegroup consisting of N-sulfo-, N-hydrocarbyl-sulfonyl- andN-hydrocarbyloxy-sulfonyl-N,N'-dilyso-gangliosides and N'-acylderivatives thereof, N'-sulfo-, N'hydrocarbyl-sulfonyl- andN'-hydrocarbyloxy-sulfonyl-N,N'-dilyso-gangliosides and N-acylderivatives thereof; N,N'-di or polysulfo-N,N'-di-orpoly-lyso-gangliosides, N,N'-di or polyhydrocarbylsulfonyl-N,N'-di- orpoly-lyso-gangliosides and N,N'-di- or polyhydrocarbyloxy-N,N'-di- orpoly-lyso-gangliosides; salts of said ganglioside analogues; esters andamides of the carboxyl groups of the sialic acid residue portion of saidganglioside analogues; inner esters of the sialic acid carboxyl groupsand the oligosaccharide hydroxyl groups of said ganglioside analogues;and organic or sulfonic acid esters of the hydroxyl groups of saidganglioside analogues.
 2. Ganglioside analogues according to claim 1,wherein the hydrocarbyl radical is selected from the group consisting ofalkyl with up to 24 carbon atoms, aryl with up to 24 carbon atoms,aralkyl with up to 24 carbon atoms, and cycloalkyl with up to 24 carbonatoms.
 3. Ganglioside analogues according to claim 2, whereinhydrocarbyl is an alkyl radical with a maximum of 24 carbon atoms. 4.Ganglioside analogues according to claim 3, wherein the alkyl radical issubstituted with functions selected from the group consisting ofhydroxyl, amino and halogen.
 5. Ganglioside analogues according to claim4, wherein the alkyl radical is interrupted in the carbon atom chain byheteroatoms.
 6. Ganglioside analogues according to claim 3, wherein thealkyl radical has a maximum of 6 carbon atoms.
 7. Ganglioside analoguesaccording to claim 3, wherein the alkyl radical is saturated orunsaturated and has between 14 and 22 carbon atoms.
 8. Gangliosideanalogues according to claim 2, wherein the aryl radical has a maximumof 12 carbon atoms.
 9. Ganglioside analogues according to claim 8,wherein the aryl radical is an unsubstituted phenyl group or a phenylgroup substituted with 1 to 3 C₁₋₄ alkyl groups.
 10. Gangliosideanalogues according to claim 2, wherein the aralkyl radical has amaximum of 12 carbon atoms.
 11. Ganglioside analogues according to claim10, wherein the aralkyl radical is formed by a C₂₋₄ alkylene group andan aromatic portion formed by a phenyl group unsubstituted orsubstituted with 1 to 3 C₁₋₄ alkyl groups or C₁₋₄ alkoxy groups. 12.Ganglioside analogues according to claim 2, wherein the cycloalkylradical is selected from the group consisting of cyclopropyl,cyclohexyl, cyclobutyl, and cyclopentyl, wherein said cycloalkyl radicalmay be optionally substituted with C₁₋₄ alkyl groups.
 13. Gangliosideanalogues according to claim 1 excluding N'-acyl derivatives and N-acylderivatives.
 14. Ganglioside analogues according to claim 1, wherein theacyl group on the sphingosine N or on the neuraminic N' is from analiphatic acid with a maximum of 24 carbon atoms.
 15. Gangliosideanalogues according to claim 14, wherein the acyl group is substitutedwith a polar unit selected from the group consisting of halogens, freeor esterified hydroxy-L groups, and free or esterified mercaptan groups.16. Ganglioside analogues according to claim 1, wherein the acyl groupon the sphingosine N or on the neuraminic N' is from benzoic acid or abenzoic acid homoloque where the phenyl group is substituted with 1 to 3substituents selected from the group consisting of C₁₋₄ alkyl, C₁₋₄alkoxyl, amino, C₁₋₄ -alkylamino and di(C₁₋₄ alkyl)amino. 17.Ganglioside analogues according to claim 1, wherein the acyl group onthe sphingosine N or on the neuraminic N' is from an araliphatic acidwith a C₂₋₄ aliphatic-alkylene chain and an aromatic portion wherein thephenyl group is substituted with 1-3 substituents selected from thegroup consisting of C₁₋₄ alkyl, C₁₋₄ alkoxy, amino, C₁₋₄ alkylamino anddi(C₁₋₄ alkyl)amino.
 18. Ganglioside analogues according to claim 1,wherein the acyl group on the sphingosine N or on the neuraminic N' isfrom cyclopropane, cyclobutane, cyclopentane or cyclohexane carboxylicacid.
 19. Ganglioside analogues according to claim 1, wherein the acylgroup on the sphingosine N or on the neuraminic N' is from aheterocyclic acid with a single heterocyclic ring, wherein saidheterocyclic ring contains a single heteroatom selected from the groupconsisting of O, N and S and wherein said heterocyclic ring is of eitheraromatic or aliphatic nature.
 20. Ganglioside analogues according toclaim 1 which are carboxyl esters from an aliphatic alcohol with amaximum of 12 carbon atoms, or from an araliphatic alcohol with abenzene ring unsubstituted or substituted with 1 to 3 C₁₋₄ alkyl groups,and having a maximum of 4 carbon atoms in the aliphatic portion of thearaliphatic alcohol.
 21. Ganglioside analogues according to claim 1which are carboxyl amides from ammonium or an aliphatic amine with amaximum of 12 carbon atoms.
 22. Ganglioside analogues according to claim1 esterified with sulfonic acid.
 23. Ganglioside analogues according toclaim 1 in the form of a mixture, wherein a first analogue is esterifiedwith sulfuric acid forming an esterified hydroxyl, and wherein a secondanalogue is not esterified with sulfuric acid.
 24. The functionalderivative according to claim 22 which is a total ester.
 25. Gangliosideanalogues according to claim 1 esterified with an organic acid. 26.Ganglioside analogues according to claim 25, in which the ester is aperacylated moiety wherein the acyl group is from an aliphatic acid witha maximum of 24 carbon atoms or from an araliphatic acid in which thealiphatic moiety is a C₂₋₄ alkylene group and the phenyl moiety isoptionally substituted with 1 to 3 methyl or methoxyl groups.
 27. Acompound selected from the group consistingof:N-ethyl-sulfonyl-N'-acetyl-N,N'-dilyso-GM₁,N-propyl-sulfonyl-N'-acetyl-N,N'-dilyso-GM₁,N-n-butyl-sulfonyl-N'-acetyl-N,N'-dilyso-GM₁,N-n-pentyl-sulfonyl-N'-acetyl-N,N'-dilyso-GM₁,N-n-hexyl-sulfonyl-N'-acetyl-N,N'-dilyso-GM₁,N-n-heptyl-sulfonyl-N'-acetyl-N,N'-dilyso-GM₁,N-n-octyl-sulfonyl-N'-acetyl-N,N'-dilyso-GM₁,N-n-decyl-sulfonyl-N'-acetyl- N,N'-dilyso- GM₁,N-2-bromoethyl-sulfonyl-N'-acetyl-N,N'-dilyso-GM₁,N-hexadecyl-sulfonyl-N'-acetyl-N,N'-dilyso-GM₁,N-3-chloropropyl-sulfonyl-N'-acetyl-N,N'-dilyso-GM₁,N-6-bromohexyl-sulfonyl-N'-acetyl-N,N'-dilyso-GM₁,N-benzyl-sulfonyl-N'-acetyl-N,N'-dilyso-GM₁,N-4-chlorobenzyl-sulfonyl-N'-acetyl-N,N'-dilyso-GM₁,N-4-aminobenzyl-sulfonyl-N'-acetyl-N,N'-dilyso-GM₁,N-3,4,5-trimethoxybenzyl-sulfonyl-N'-acetyl-N,N'-dilyso-GM₁, andN-sulfo-N'-acetyl-N,N'-dilyso-GM₁.
 28. A compound selected from thegroup consisting of N'-sulfo-N'-lyso-GM₁ and esters thereof, whereinsaid esters are from alcohols selected from the group consisting ofethyl alcohol, propyl alcohol, n-butyl alcohol, n-pentyl alcohol,n-hexyl alcohol, n-heptyl alcohol, n-octyl alcohol, n-decyl alcohol,2-bromoethyl alcohol, hexadecyl alcohol, 3-chloropropyl alcohol,6-bromohexyl alcohol, benzylsulfonyl alcohol, 4-chlorobenzyl alcohol,4-aminobenzyl alcohol, and 3,4,5-trimethoxy-benzyl alcohol.
 29. Acompound selected from the group consisting ofN,N'-di-sulfo-N,N'-di-lyso-GM₁ and esters thereof, wherein said estersare from alcohols selected from the group consisting of ethyl alcohol,propyl alcohol, n-butyl alcohol, n-pentyl alcohol, n-hexyl alcohol,n-heptyl alcohol, n-octyl alcohol, n-decyl alcohol, 2-bromoethylalcohol, hexadecyl alcohol, 3-chloropropyl alcohol, 6-bromohexylalcohol, benzyl-sulfonyl alcohol, 4-chlorobenzyl alcohol, 4-aminobenzylalcohol, and 3,4,5-trimethoxybenzyl alcohol.
 30. A compound selectedfrom the group consisting of: N-ethyl-sulfonyl-N'-acyl-N,N'-dilyso-GM₁,N-propyl sulfonyl-N'-acyl-N,N'-dilyso-GM₁,N-n-butyl-sulfonyl-N'-acyl-N,N'-dilyso-GM₁,N-n-pentyl-sulfonyl-N'-acyl-N,N'-dilyso-GM₁,N-n-hexyl-sulfonyl-N'-acyl-N,N'-dilyso-GM₁,N-n-octyl-sulfonyl-N'-acyl-N,N'-dilyso-GM₁,N-n-decyl-sulfonyl-N'-acyl-N,N'-dilyso-GM₁,N-2-bromoethyl-sulfonyl-N'-acyl-N,N'-dilyso-GM₁,N-hexadecyl-sulfonyl-N'-acyl-N,N'-dilyso-GM₁,N-3-chloropropyl-sulfonyl-N'-acyl-N,N'-dilyso-GM₁,N-6-bromohexyl-sulfonyl-N'-acyl-N,N'-dilyso-GM₁,N-benzyl-sulfonyl-N'-acyl-N,N'-dilyso-GM₁,N-4-chlorobenzyl-sulfonyl-N'-acyl-N,N'-dilyso-GM₁,N-4-aminobenzyl-sulfonyl-N'-acyl-N,N'-dilyso-GM₁,N-3,4,5-trimethoxybenzyl-sulfonyl-N'-acyl-N,N'-dilyso-GM₁,N-acyl-N'-ethyl-sulfonyl-N,N'-dilyso-GM₁, N-acyl-N'-propylsulfonyl-N,N'-dilyso-GM₁, N-acyl-N'-n-butyl-sulfonyl-N,N'-dilyso-GM₁,N-acyl-N'-pentyl-sulfonyl-N,N'-dilyso-GM₁,N-acyl-N'-n-hexyl-sulfonyl-N,N'-dilyso-GM₁,N-acyl-N'-n-heptyl-sulfonyl-N,N'-dilyso-GM₁,N-acyl-N'-n-octyl-sulfonyl-N,N'-dilyso-GM₁,N-acyl-N'-n-decyl-sulfonyl-N,N'-dilyso-GM₁,N-acyl-N'-2-bromoethyl-sulfonyl-N,N'-dilyso-GM₁,N-acyl-N'-hexadecyl-sulfonyl-N,N'-dilyso-GM₁,N-acyl-N'-3-chloropropyl-sulfonyl-N,N'-dilyso-GM₁,N-acyl-N'-6-bromohexyl-sulfonyl-N,N'-dilyso-GM1,N-acyl-N'-benzyl-sulfonyl-N,N'-dilyso-CM₁,N-acyl-N-4-aminobenzyl-sulfonyl-N,N'-dilyso-GM₁, andN-acyl-N'-3,4,5-trimethoxybenzyl-sulfonyl-N,N'-dilyso-GM₁, wherein theacyl group is from an acid selected from the group consisting of acetic,chloroacetic, dichloroacetic, propionic, valeric, trimethyl-acetic acid,caproic acid, and capric acid.
 31. Pharmacologically acceptable salts ofany one of the analogues as claimed in claim 1, wherein the cation ofthe salt is a metallic or basic cation.
 32. The salt according to claim31 which is a sodium salt.
 33. Pharmaceutical compositions comprising atleast one compound according to claim 1 and a pharmaceuticallyacceptable excipient, for use in therapy.
 34. A method of reducingglutamate-induced neurotoxicity which comprises administering to apatient in need thereof a pharmaceutical composition according to claim33.
 35. A method for inhibiting expression of CD₄ determinants presenton the surface of human cells which comprises administering to saidcells a pharmaceutical composition according to claim
 33. 36. A processfor the preparation of ganglioside analogues according to claim 1, whichcomprises treating a N,N'-di-lyso-ganglioside or a derivative or a saltthereof with sulfuric acid, a hydrocarbyloxy-sulfuric acid or ahydrocarbyl-sulfonic acid, or a derivative thereof, optionally acylatingan amino group in position N- or N'-, and in the resulting acylatedamino groups optionally hydrolyzing one of the two acylated amino groupsinto free amino groups, optionally converting free carboxyl groups orfree hydroxyl groups into their functional derivatives, and optionallyconverting obtained compounds into corresponding metal, organic base oracid salts.
 37. Compounds with the following formula: ##STR5## wherein:R=H or SO₃ H;R₁ =H, ##STR6## R₂ =--(CH₂)_(n) --CH₃, wherein n=12-14; R₃=H, acyl or SO₂ R₅, provided that at least one R₃ is SO₂ R₅, R₄ =H, or##STR7## R₅ =alkyl, aryl or OX, wherein X=H, alkyl or aryl.